Process for producing arsenic trioxide formulations and methods for treating cancer using arsenic trioxide or melarsoprol

ABSTRACT

The invention relates to the use of arsenic compounds to treat a variety of leukemia, lymphoma and solid tumors. Further, the arsenic compounds may be used in combination with other therapeutic agents, such as a retinoid. The invention also provides a process for producing arsenic trioxide formulations.

1. FIELD OF INVENTION

The present invention relates to methods and compositions for thetreatment of leukemia, lymphoma, and certain other cancers.

More specifically, the present invention relates to the novel uses ofarsenic trioxide and an organic arsenic compound for treating acuteleukemia and chronic leukemia.

2. BACKGROUND OF THE INVENTION

2.1. Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth as exemplified by hyperplasia,metaplasia, and dysplasia (for review of such abnormal growthconditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B.Saunders Co., Philadelphia, pp. 68-79) precedes the formation of aneoplastic lesion. A neoplastic lesion may evolve clonally to grow intoa solid tumor, and develop an increasing capacity for invasion, growth,metastasis, and heterogeneity, especially under conditions in which theneoplastic cells escape the host's immune surveillance (Roitt, I.,Brostoff, J and Kale, D., 1993, Immunology, 3rd ed., Mosby, St. Louis,pps. 17.1-17.12).

Leukemia refers to malignant neoplasms of the blood-forming tissues.Transformation to malignancy typically occurs in a single cell throughtwo or more steps with subsequent proliferation and clonal expansion. Insome leukemias, specific chromosomal translocations have been identifiedwith consistent leukemic cell morphology and special clinical features(e.g., translocations of 9 and 22 in chronic myelocytic leukemia, and of15 and 17 in acute promyelocytic leukemia). Acute leukemias arepredominantly undifferentiated cell populations and chronic leukemiasmore mature cell forms.

Acute leukemias are divided into lymphoblastic (ALL) andnon-lymphoblastic (ANLL) types. They may be further subdivided by theirmorphologic and cytochemical appearance according to theFrench-American-British (FAB) classification or according to their typeand degree of differentiation. The use of specific B- and T-cell andmyeloid-antigen monoclonal antibodies are most helpful forclassification. ALL is predominantly a childhood disease which isestablished by laboratory findings and bone marrow examination. ANLL,also known as acute myeloblastic leukemia (AML), occurs at all ages andis the more common acute leukemia among adults; it is the form usuallyassociated with irradiation as a causative agent.

Chronic leukemias are described as being lymphocytic (CLL) or myelocytic(CML). CLL is characterized by the appearance of mature lymphocytes inblood, bone marrow, and lymphoid organs. The hallmark of CLL issustained, absolute lymphocytosis (>5,000/μL) and an increase oflymphocytes in the bone marrow. Most CLL patients also have clonalexpansion of lymphocytes with B-cell characteristics. CLL is a diseaseof older persons. In CML, the characteristic feature is the predominanceof granulocytic cells of all stages of differentiation in blood, bonemarrow, liver, spleen, and other organs. In the symptomatic patient atdiagnosis the total WBC count is usually about 200,000/μL, but may reach1,000,000/μL. CML is relatively easy to diagnose because of the presenceof the Philadelphia chromosome.

The very nature of hematopoietic cancer necessitates using systemicchemotherapy as the primary treatment modality. Drugs selected accordingto sensitivities of specific leukemias are usually given in combination.Radiation therapy may be used as an adjunct to treat local accumulationsof leukemic cells. Surgery is rarely indicated as a primary treatmentmodality, but may be used in managing some complications. Bone marrowtransplantation from an HLA-matched sibling is sometimes indicated.

2.2. Arsenic and its Medical Uses

Arsenic has been considered to be both a poison and a drug for a longtime in both Western and Chinese medical practices. In the latter partof the nineteenth century, arsenic was used frequently in attempts totreat diseases of the blood in the West. In 1878, it was reported thattreatment of a leukemic patient with Fowler's solution (a solutioncontaining potassium arsenite, valence +5) reduced markedly the count ofwhite blood cells (Cutler and Bradford, Am. J. Ned. Sci., January 1878,81-84). Further interests in the use of Fowler's solution as apalliative agent to treat chronic myelogenous leukemia (CML) wasdescribed by Forkner and Scott in 1931 (J. Am. Med. Assoc., 1931, iii,97), and later confirmed by Stephens and Lawrence in 1936 (Ann. Intern.Med. 9, 1488-1502). However, while the active chemical ingredient(s) ofFowler's solution was not determined, its toxicity was well recognized.Fowler's solution was administered strictly as an oral composition, andwas given to leukemic patients as a solution until the level of whiteblood cells was depressed to an acceptable level or until toxicities(such as skin keratoses and hyperpigmentation) developed, while thepatients enjoyed varying periods of remission. In the 1960's, Fowler'ssolution was still used occasionally in attempts to treat CML, however,most patients with CML were treated with other chemotherapeutic agents,such as busulfan, and/or radiation therapy (Monfardini et al., Cancer,1973, 31:492-501).

Paradoxically, one of the long recognized effects of exposure toarsenic, whether the source is environmental or medicinal, is skincancer (Hutchinson, 1888, Trans. Path. Soc. Lond., 39:352; Neubauer,1947, Br. J. Cancer, 1:192). There were even epidemiological data tosuggest that the use of Fowler's solution over long periods could leadto an increased incidence of cancer at internal sites (Cuzick et al.,Br. J. Cancer, 1982, 45:904-911; Kaspar et al., J. Am. Med. Assoc.,1984, 252:3407-3408). The carcinogenicity of arsenic has since beendemonstrated by the fact that it can induce chromosomal aberration, geneamplification, sister chromatid exchanges and cellular transformation(See e.g., Lee et al., 1988, Science, 241:79-81; and Germolec et al.,Toxicol. Applied Pharmacol., 1996, 141:308-318). Because of the knowncarcinogenic effect of arsenic, its only therapeutic use in human inWestern medicine today is in the treatment of tropical diseases, such asAfrican trypanosomiasis, (the organic arsenical, melarsoprol; SeeGoodman & Gilman's The Pharmacological Basis of Therapeutics, 9thedition, chapter 66, 1659-1662, 1997).

In traditional chinese medicine, arsenous acid or arsenic trioxide pastehas been used to treat tooth marrow diseases, psoriasis, syphilis andrheumatosis (Chen et al., 1995, in Manual of Clinical Drugs, Shanghai,China, Shanghai Institute of Science and Technology, p.830). In 1970's,arsenic trioxide had been applied experimentally to treat acutepromyelocytic leukemia (APL) in China (commented by Mervis, 1996,Science, 273:578). The clinical efficacy of arsenic trioxide hasrecently been re-investigated in 14 of 15 patients with refractory APL,where the use of an intravenous dose at 10 mg/day for 4-9 weeks wasreported to result in complete morphologic remission without associatedbone marrow suppression (Shen et al., 1997, Blood, 89:3354-3360). It wasalso shown that arsenic trioxide induced apoptosis (programmed celldeath) in vitro in NB4 cells, an APL cell line, and that apoptosis wasapparently associated with down-regulation of the oncogene bcl-2, andintracellular redistribution of the chimeric PML/RARα protein that areunique to APL cells (Chen et al., 1996, Blood, 88:1052-1061; Andre etal., 1996, Exp. Cell Res. 229:253-260). It has been reported that thebiological activity of arsenic is due to the ability of arsenic todirect the nucleoplasmic fraction of PML to nuclear bodies fordegradation (Zhu et al., 1997, Proc. Natl. Acad. Sci., 94:3978-3983).

Although arsenic is well known to be both a poison and a carcinogenicagent, there have been many reports concerning the use of arsenic inmedical treatment. Further, from the above discussion, it should beclear that there are many different types of leukemias, each of whichrequires a unique treatment protocol that is modified according to thepresence of factors predicting for a risk of treatment failure. Thus,the development of a broad spectrum anti-leukemia agent that can be usedalone or in combination with other existing drugs is extremelydesirable.

3. SUMMARY OF THE INVENTION

Despite the conflicting reports in the art concerning benefits and risksof the administration of arsenic to patients, applicants have discoveredthat arsenic trioxide and the organic arsenical, melarsoprol, have broadapplicability in the treatment of various types of leukemias, lymphomas,and solid tumors.

The invention described herein encompasses a method of treatingleukemia, lymphoma or solid tumors comprising the administration of atherapeutically effective and non-lethal amount of arsenic trioxide ormelarsoprol to a human in need of such therapy. The invention, asmentioned above also encompasses the use of combination therapy to treatleukemia, especially leukemias which are refractory to other forms oftreatment.

The invention also encompasses a method for the manufacture ofpharmaceutical compositions comprising arsenic trioxide.

In accordance with the present invention, arsenic trioxide ormelarsoprol compounds can be used alone or in combination with otherknown therapeutic agents (including chemotherapeutics, radioprotectantsand radiotherapeutics) or techniques to either improve the quality oflife of the patient, or to treat leukemia, lymphoma or solid tumor. Thearsenic compounds can be used before, during or after the administrationof one or more known chemotherapeutic agents, including antitumoragents. In addition, the arsenic compounds can be used before, during orafter radiation treatment.

The pharmaceutical compositions of the invention are sterile solutionssuitable for intravenous injection or infusion. In another embodimentthe invention encompasses a composition suitable for oral delivery;comprising arsenic trioxide or melarsoprol and a pharmaceuticallyacceptable excipient or carrier. In another embodiment, the inventionalso includes compositions suitable for topical or transdermal delivery,including but not limited to iontophoretic methods. Specific therapeuticregimens, pharmaceutical compositions, and kits are also provided by theinvention.

Particular compositions of the invention and their uses are described inthe sections and subsections which follow.

4. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the treatment of leukemia, lymphoma orsolid tumors are described herein. This invention provides a method oftreating acute or chronic leukemia, lymphoma, or solid tumors in a humanwhich comprises administering to a human in need of such therapy atherapeutically effective and non-lethal amount of one or more arseniccompounds, such as arsenic trioxide or melarsoprol.

The invention also includes a method of treating leukemia in a human whohas become refractory to other forms of treatment which comprisesadministering to a human arsenic trioxide or melarsoprol in combinationwith another chemotherapeutic agent, e.g., all-trans retinoic acid(ATRA).

The invention also relates to a method for the manufacture ofpharmaceutical compositions comprising arsenic trioxide. It is preferredthat pharmaceutical compositions of the present invention exhibitreduced toxicity, improved efficacy, improved stability during storageand use, and that the composition has a physiologically acceptable pH.

4.1. The Arsenic Compounds

As used herein, “arsenic compound” refers to a pharmaceuticallyacceptable form of arsenic trioxide (As₂O₃) or melarsoprol. Melarsoprolis an organic arsenic compound which can be synthesized by complexingmelarsen oxide with dimercaprol or commercially purchased (Arsobal® byRhone Poulenc Rorer, Collegeville, Pa.). Since the non-pharmaceuticallyformulated raw materials of the invention are well known, they can beprepared from well-known chemical techniques in the art. (See forexample, Kirk-Othmer, Encyclopedia of Chemical Technology 4th ed. volume3 pps. 633-655 John Wiley & Sons).

As used herein the terms “a therapeutic agent”, “therapeutic regimen”,“radioprotectant”, “chemotherapeutic” mean conventional drugs and drugtherapies, including vaccines, for treating cancer, viral infections,and other malignancies, which are known to those skilled in the art.“Radiotherapeutic” agents are well known in the art.

In accordance with the present invention, arsenic trioxide ormelarsoprol compounds can be used alone or in combination with otherknown therapeutic agents (including chemotherapeutics, radioprotectantsand radiotherapeutics) or techniques to either improve the quality oflife of the patient, or to treat leukemia, lymphoma or solid tumor. Forexample, the arsenic compounds can be used before, during or after theadministration of one or more known antitumor agents including but notlimited to mustard compounds, nitrogen mustard, chlorambucil, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil, floxuridine, methotrexate, vincristine, vinblastine,taxol, etoposide, temiposide, dactinomycin, daunorubicin, doxorubicin,bleomycin, mitomycin, cisplatin, carboplatin, estramustine phosphate,hydroxyurea, BCNU, procarbazine, VM-26, interferons, and all-transretinoic acid (ATRA), or other retinoids (See, for example, thePhysician Desk References 1997). In addition, the arsenic compounds canbe used before, during or after radiation treatment.

In a specific embodiment, the arsenic compound of the invention and ATRAcan be administered as a mixture. In preferred aspects, the lymphoma,leukemia or solid tumor in the human treated by the combination isrefractory to general methods of treatment, or is a relapsed case ofleukemia.

Any suitable mode of administration may be used in accordance with thepresent invention including but not limited to parenteral administrationsuch as intravenous, subcutaneous, intramuscular and intrathecaladministration; oral, and intranasal administration, and inhalation. Themode of administration will vary according to the type of cancer, andthe condition of the human.

The pharmaceutical compositions to be used may be in the form of sterileaqueous or organic solutions; colloidal suspensions, caplets, tabletsand cachets.

4.2. Methods Of Treatment

The term “a method for treating leukemia” as used herein means that thedisease and the symptoms associated with the disease are alleviated,reduced, cured, or placed in a state of remission. For example, themethods of treatment of the invention can lower the white blood cellcount, or reduce lymphocytosis in a human under treatment.

The term “a method for treating lymphoma” as used herein means that thedisease and the symptoms associated with the disease are alleviated,reduced, cured, or placed in a state of remission.

The term “a method for treating solid tumor” as used herein means thatthe disease and the symptoms associated with the solid tumor arealleviated, reduced, cured, or placed in a state of remission.

In addition, the term “a method for treating leukemic infiltration”means that the infiltration of leukemic cells out of circulation andinto other organs and systems and the symptoms associated with suchinfiltration are alleviated, reduced, cured, or placed in a state ofremission.

The term “refractory” when used herein means that the leukemia isgenerally resistant to treatment or cure.

As used herein, “preneoplastic” cell refers to a cell which is intransition from a normal to a neoplastic form; or cells that fail todifferentiate normally; and morphological evidence, increasinglysupported by molecular biologic studies, indicates that preneoplasiaprogresses through multiple steps.

In one embodiment, the invention provides a method for treatment ofleukemia in a human comprising the administration of a therapeuticallyeffective and non-lethal amount of arsenic trioxide or melarsoprol tothe human. The invention also provides a weight-based dosing regimen,not heretofore disclosed, that maximizes the safety in humans of theseotherwise highly toxic compounds.

Arsenic trioxide (As₂O₃) inhibits growth and induce apoptosis in NB4acute promyelocytic leukemic cells. Acute promyelocytic leukemia (APL)is associated with the t(15;17) translocation, which generates aPML/RARα fusion protein between PML, a growth suppressor localized onnuclear matrix-associated bodies, and RARα, a nuclear receptor forretinoic acid (RA). PML/RARα was proposed to block myeloiddifferentiation through inhibition of nuclear receptor response, as doesa dominant negative RARα mutant. In addition, in APL cells, PML/RARαdisplaces PML and other nuclear body (NB) antigens onto nuclearmicrospeckles, likely resulting in the loss of PML and/or NB functions.It has been suggested that high concentrations of arsenic trioxidepromote apoptosis, whereas low concentrations induce partialdifferentiation in NB4 cells as well as cells derived from APL patients.It was postulated that As₂O₃ works through its ability to specificallycause PML-RARα in APL cells to be relocalized to nuclear bodies fordegradation (Zhu et al., 1997, Proc. Natl. Acad. Sci. USA,94:3978-3983). However, these findings tend to limit the use of arsenictrioxide to a subset of leukemias. See Konig et al., 1997, Blood,90:562-570.

Unexpectedly, the inventors have discovered that both As₂O₃ andmelarsoprol are able to inhibit cell growth, and induce apoptosis invarious myeloid leukemia cell lines in a PML and PML-RARα independentmanner. Thus, the inventors have discovered that, contrary to theearlier findings, arsenic trioxide and melarsoprol are both effectiveagainst a broad range of leukemias regardless of the underlyingmolecular mechanism that causes the neoplasia. Working examples of theeffect of arsenic compounds on a number of leukemic cell lines areprovided in Sections 5.1 and 5.2.

Accordingly, the arsenic compounds of the present invention can be usedagainst a variety of leukemias, including but not limited to;

-   -   Acute lymphoblastic leukemia (ALL)    -   Acute lymphoblastic B-cell leukemia    -   Acute lymphoblastic T-cell leukemia    -   Acute myeloblastic leukemia (AML)    -   Acute promyelocytic leukemia (APL)    -   Acute monoblastic leukemia    -   Acute erythroleukemic leukemia    -   Acute megakaryoblastic leukemia    -   Acute myelomonocytic leukemia    -   Acute undifferentiated leukemia    -   Chronic myelocytic leukemia (CML)    -   Chronic lymphocytic leukemia (CLL)

The skilled artisan will recognize that other leukemias may be treatedin accordance with the present invention.

In another embodiment, the invention provides a method for treatment oflymphoma in a human comprising the administration of a therapeuticallyeffective and non-lethal amount of arsenic trioxide or melarsoprol tothe human. Lymphoma that can be treated by the methods of the inventioninclude but are not limited to high grade lymphoma, intermediate gradelymphoma, low grade lymphoma, and the various subclassifications.

In yet another embodiment, the invention provides a method for treatmentof solid tumors, including metastasises, in humans comprising theadministration of a therapeutically effective and non-lethal amount ofarsenic trioxide or melarsoprol to the human. Solid tumors that can betreated by the methods of the invention include but are not limited to:cancer of the digestive tract, oesophagus, liver, stomach, and colon;skin; brain; bone; breast; lung; and soft tissues, including but notlimited to various sarcomas, and preferably prostate cancer.

In various embodiments, the leukemic or tumor cells are infiltratingother organs and systems in a human, for example, the central nervoussystem. The methods of the invention are also applicable to reduce thenumber of preneoplastic cells in a human in which there is an abnormalincrease in the number of preneoplastic cells.

In a specific embodiment, the invention provides a method of treatmentof acute promyelolytic leukemia (APL) in a human comprising theadministration of a therapeutically effective and non-lethal amount ofmelarsoprol to the human. The inventors discovered, as described inSection 5.2, that concentrations of melarsoprol that are cytotoxic invitro can readily be achieved in vivo.

In one specific embodiment, the invention provides a method of treatmentof chronic myelogenous leukemia (CML) in a human comprising theadministration of a therapeutically effective and non-lethal amount ofarsenic trioxide to the human. The inventors discovered, as described inSection 5.3, that arsenic trioxide can also induce apoptosis in a CMLcell line. The therapeutic benefits of the pharmaceutical compositionsof the invention comprising arsenic trioxide is far superior to that ofpotassium arsenite, commonly formulated as Fowler's solution.

In yet another specific embodiment, the invention provides a method oftreatment of acute promyelocytic leukemia (APL) in a human, in which theAPL is associated with a translocation of the RARα locus on chromosome17 to chromosome 11, comprising the administration of a therapeuticallyeffective amount of arsenic trioxide or melarsoprol to the human. In themajority of APL cases, RARα on chromosome 17 translocates and fuses withthe PML gene located on chromosome 15, i.e., t(15;17). In a few casesRARA translocates to chromosome 11 where it fuses to the PLZF gene.Patients harboring the t(15;17) are uniquely sensitive to treatment withall-trans retinoic acid (ATRA), yielding complete remission rates of 75%to 95%. APL associated with the t(11;17) (PLZF-RARa) shows a distinctlyworse prognosis with poor response to chemotherapy and little or noresponse to treatment with ATRA, thus defining a new APL syndrome. Thepresent invention provides that arsenic trioxide or melarsoprol can beused to treat such cases of APL. Transgenic animal models of APLassociated with t(15;17) and t(11;17) for testing the therapeuticbenefits and dosages of arsenic compounds of the invention are describedin Section 5.4 hereinbelow.

Humans having leukemia are sometimes refractory to conventional methodsof treatment by reason of having undergone anti-leukemic therapy (e.g.,chemotherapy). Thus, the invention provides a method of treatment ofleukemia in a human who is not responding to conventional therapycomprising the administration of a therapeutically effective andnon-lethal amount of a combination of arsenic compound and anotherchemotherapeutic agent, such as but not limited to, all-trans retinoicacid (ATRA) or other retinoids, to the human. The arsenic compound caneither be arsenic trioxide or melarsoprol or a pharmaceuticallyacceptable salt thereof. The invention also encompasses the treatment ofretinoid-resistant patients with an arsenic compound.

In specific embodiments, the arsenic compound of the invention and thechemotherapeutic agent can be administered either as a mixture orsequentially. When administered sequentially, the arsenic compound maybe administered before or after the chemotherapeutic agent, so long asthe first administered agent is still providing antileukemic activity inthe human when the second agent is administered. Any of the modes ofadministration described herein may be used to deliver the combination.In preferred aspects, the leukemia in the human treated by thecombination is refractory to general methods of treatment, or is arelapsed case of leukemia.

4.3. Process for The Manufacture Of Sterile Arsenic Trioxide Solution

The arsenic compounds of the invention may be formulated into sterilepharmaceutical preparations for administration to humans for treatmentof leukemias, lymphomas and solid tumors. Compositions comprising acompound of the invention formulated in a compatible pharmaceuticalcarrier may be prepared, packaged, labelled for treatment of and usedfor the treatment of the indicated leukemia, lymphoma, or solid tumor.

In one aspect, the invention provides a method for the manufacture of apharmaceutical composition comprising a therapeutic effective andnon-lethal amount of arsenic trioxide (As₂O₃). Arsenic trioxide (rawmaterial) is a solid inorganic compound that is commercially availablein a very pure form. However, it is difficult to dissolve As₂O₃ inaqueous solution. Further, the inventors are unaware of any publishedteachings on how to formulate As₂O₃ as a pharmaceutical compositionsuitable for injection directly into a human. Arsenic is present insolution in the +5 valence state (pentavalent) or the +3 valence state(trivalent). For example, potassium arsenite (KAsO₂; which is present inFowler's solution) and salts of arsenious acid contain pentavalentarsenic. It is known that one form of arsenic is more toxic than theother. (Goodman & Gilman's The Pharmacological Basis of Therapeutics,9th edition, chapter 66, 1660, 1997). A fresh solution of arsenictrioxide containing arsenic in the trivalent state will be graduallyoxidized to pentavalent state if exposed to air for a prolonged period,and as a result of the accumulation of pentavalent arsenic, the relativetoxicity of a solution of As₂O₃ will change over time. (Id.)Furthermore, it is observed that the total amount of arsenic in solutiondecreases over time. This loss of material is caused by the progressiveconversion of arsenic in the solution into arsine (AsH₃) which is agaseous compound at room temperature. This is particularly problematicin pharmaceutical applications if the concentration of an activeingredient in the injected material cannot be controlled. It is alsoundesirable to allow arsine to escape from the solution into theatmosphere because arsine is also toxic.

The inventors have experimented and successfully developed a method forformulating arsenic trioxide which overcomes the above-describedproblems of solubility and stability. The method comprises solubilizingsolid high purity As₂O₃ in an aqueous solution at high pH, such as pHgreater than 12. For example, a 5 M solution of sodium hydroxide may beused. To aid solubilization and obtain a clear and homogenous solution,mechanical stirring and/or gentle heating may be applied. A solution ofAs₂O₃ can also be obtained by dissolving the solid compound overnight.Typically, a solution of 1 M As₂O₃ is obtained by this method. However,this solution is too basic to be useful as a pharmaceutical composition.

To adjust the pH of the As₂O₃ solution, the solution is first diluted inwater, for example, to a concentration of about 1 mg/mL, pH 12. TheAs₂O₃ solution is then back-titrated with acid, such as, hydrochloricacid (1 M to 5 M HCl), with constant stirring until the pH is about 8.0to 8.5. Highly concentrated HCl is not suitable as it causesprecipitation to occur in the As₂O₃ solution. The partially neutralizedAs₂O₃ solution may then be sterilized for example, by filtration (e.g.,through a 0.22 μm filter), and stored in sterile vials.

To make a pharmaceutical composition that can be directly injected intoa subject, the composition must be sterile, standard techniques known tothe skilled artisan for sterilization can be used. See, e.g.,Remington's Pharmaceutical Science. The pH of the partially neutralizedAs₂O₃ solution may be further adjusted to near physiological pH bydilution (10-100 fold) with a pharmaceutical carrier, such as a 5%dextrose solution. For example, 10 mL of a partially neutralized As₂O₃solution can be added to 500 mL of a 5% dextrose solution to yield afinal pH of about 6.5 to 7.5. The method of the invention reduces theoxidation of arsenic in solution. Pharmaceutical compositions containingarsenic trioxide manufactured by the method of the invention showimproved stability and long shelf life.

4.4. Pharmaceutical Composition and Modes of Administration

According to the invention, the arsenic compounds and theirphysiologically acceptable solvates may be formulated for oral orparenteral administration.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or is continuous infusion.Formulations for injection may be presented in unit dosage form, e.g.,in ampules or in multi-dose containers, with an added preservative. Thecompositions may take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively, theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically effective amounts of the arsenic compounds inpharmaceutically acceptable form. The arsenic compound in a vial of akit of the invention may be in the form of a pharmaceutically acceptablesolution, e.g., in combination with sterile saline, dextrose solution,or buffered solution, or other pharmaceutically acceptable sterilefluid. Alternatively, the complex may be lyophilized or desiccated; inthis instance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of arsenic compounds by a clinician or bythe patient.

The magnitude of a therapeutic dose of an arsenic compound in the acuteor chronic management of leukemia will vary with the severity of thecondition to be treated and the route of administration. The dose, andperhaps dose frequency, will also vary according to the age, bodyweight, condition and response of the individual patient. In general,the daily dose ranges of arsenic trioxide for the conditions describedherein are generally from about 0.05 to about 5 mg per kg body weightadministered in divided doses administered parenterally or orally ortopically. A preferred total daily dose is from about 2.5 to about 40 mgof arsenic trioxide. Preferably the arsenic trioxide formulation of theinvention is given daily for a maximum of 60 days, or until remission,followed by two to ten additional cycles, each lasting about 25 days induration. For example, depending on the body weight of a patient withacute promyelocytic leukemia, a daily dose of arsenic trioxide greaterthan or less than 10 mg can be administered. Alternatively, followingthe weight-based dosing regimen, a therapeutic effect can be obtainedwith a daily dose of arsenic trioxide less than 10 mg.

For treatment of solid tumor, a preferred dosing regimen involvesintravenous infusion of about 0.1 to about 5 mg per kg body weight perday for 5 days. This five-day treatment protocol is repeated once permonth until the tumor growth tumor is inhibited or when the tumor showssigns of regression.

As for melarsoprol, the total daily dose ranges for the conditionsdescribed herein are generally from about 0.1 to about 5 mg/kg bodyweight administered in divided doses administered parenterally or orallyor topically. A preferred total daily dose is from about 0.5 to about 4mg melarsoprol per kg body weight.

The effect of the therapy with arsenic trioxide or melarsoprol ondevelopment and progression of cancer can be monitored by any methodsknown in the art, including but not limited to determining: levels oftumor specific antigens and putative biomarkers, e.g., carcinoembryonicantigens (CEA), alpha-fetoprotein; and changes in morphology and/or sizeusing computed tomographic scan and/or sonogram.

Desirable blood levels may be maintained by a continuous infusion of anarsenic compound as ascertained by plasma levels. It should be notedthat the attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

Again, any suitable route of administration may be employed forproviding the patient with an effective dosage of an arsenic compound.For example, oral, transdermal, iontophoretic, parenteral (subcutaneous,intramuscular, intrathecal and the like) may be employed. Dosage formsinclude tablets, troches, cachet, dispersions, suspensions, solutions,capsules, patches, and the like. (See, Remington's PharmaceuticalSciences.)

The pharmaceutical compositions of the present invention comprise anarsenic compound as the active ingredient, pharmaceutically acceptablesalt thereof, and may also contain a pharmaceutically acceptablecarrier, and optionally, other therapeutic ingredients, for example alltrans retinoic acid. The term “pharmaceutically acceptable salts” refersto salts prepared from pharmaceutically acceptable non-toxic acids andbases, including inorganic and organic acids and bases.

The pharmaceutical compositions include compositions suitable for oral,mucosal routes, transdermal, iontophoretic, parenteral (includingsubcutaneous, intramuscular, intrathecal and intravenous), although themost suitable route in any given case will depend on the nature andseverity of the condition being treated.

In the case where an intravenous injection or infusion composition isemployed, a suitable dosage range for use is, e.g., from about one toabout 40 mg arsenic trioxide total daily; about 0.001 to about 10 mgarsenic trioxide per kg body weight total daily, or about 0.1 to about10 mg melarsoprol per kg body weight total daily.

In addition, the arsenic carrier could be delivered via charged anduncharged matrices used as drug delivery devices such as celluloseacetate membranes, also through targeted delivery systems such asfusogenic liposomes attached to antibodies or specific antigens.

In practical use, an arsenic compound can be combined as the activeingredient in intimate admixture with a pharmaceutical carrier accordingto conventional pharmaceutical compounding techniques. The carrier maytake a wide variety of forms depending on the form of preparationdesired for administration, e.g., oral or parenteral (including tablets,capsules, powders, intravenous injections or infusions). In preparingthe compositions for oral dosage form any of the usual pharmaceuticalmedia may be employed, e.g., water, glycols, oils, alcohols, flavoringagents, preservatives, coloring agents, and the like; in the case oforal liquid preparations, e.g., suspensions, solutions, elixirs,liposomes and aerosols; starches, sugars, micro-crystalline cellulose,diluents, granulating agents, lubricants, binders, disintegratingagents, and the like in the case of oral solid preparations e.g.,powders, capsules, and tablets. In preparing the compositions forparenteral dosage form, such as intravenous injection or infusion,similar pharmaceutical media may be employed, e.g., water, glycols,oils, buffers, sugar, preservatives and the like know to those skilledin the art. Examples of such parenteral compositions include, but arenot limited to Dextrose 5% w/v, normal saline or other solutions. Thetotal dose of the arsenic compound may be administered in a vial ofintravenous fluid, e.g., ranging from about 2 ml to about 2000 ml. Thevolume of dilution fluid will vary according to the total doseadministered. For example, arsenic trioxide supplied as a 10 ml aqueoussolution at 1 mg/ml concentration is diluted in 10 to 500 ml of 5%dextrose solution, and used for intravenous infusion over a period oftime ranging from about ten minutes to about four hours.

An exemplary course of treatment of a patient with leukemia, lymphoma,or solid cancer can involve daily administration by intravenous infusionof arsenic trioxide in an aqueous solution at a daily dose of about 0.01to 1 mg arsenic trioxide per kg of body weight of the patient.Preferably, about 0.15 mg arsenic trioxide per kg body weight per day isused. The course of treatment may continue until bone marrow remissionis observed or when side effects are becoming serious. The course oftreatment may be repeated for up to ten times over approximately 10months with a break of about 3 to 6 weeks in between courses. Thepost-remission course of treatment involves infusion of arsenic trioxideat a daily dose of about 0.15 mg per kg of body weight of the patient ona daily or weekdays-only basis for a cumulative total of 25 days.

5. EXAMPLES

Described below are examples of uses of the arsenic compounds of theinvention in treatment of various types of leukemia. Through these andother experiments the arsenic trioxide formulation of the invention werefound to be well-tolerated in humans. For example, three APL patientswere given 10 mg of the arsenic trioxide formulation of the inventiononce daily (flat dose) intravenous dose.

5.1. Arsenic Trioxide and Melarsoprol Induce Apoptosis in MyeloidLeukemia Cell Lines

The activity of As₂O₃ and melarsoprol against myeloid leukemia celllines, including the APL cell line NB4-306 (a retinoic acid-resistantcell line derived from NB4 that no longer expresses the intact PML-RARαfusion protein), HL60, KG-1, and the myelomonocytic cell line U937 wasinvestigated. To examine the role of PML in mediating arsenicalactivity, the inventors also tested these agents using murine embryonicfibroblasts (MEFs) and bone marrow (BM) progenitors in which the PMLgene had been inactivated by homologous recombination. Unexpectedly, itis found that both compounds inhibited cell growth and induced apoptosisin all cell lines tested. Melarsoprol was more potent than As₂O₃ atequimolar concentrations ranging from 10⁻⁷ to 10⁻⁵ mol/L. As₂O₃relocalized PML and PML-RARα onto nuclear bodies, which was followed byPML degradation in NB4 as well as in HL60 and U937 cell lines. Althoughmelarsoprol was more potent in inhibiting growth and inducing apoptosis,it did not affect PML and/or PML-RARα nuclear localization. Moreover,both As₂O₃ and melarsoprol comparably inhibited growth and inducedapoptosis of PML+/+ and PML−/−MEF, and inhibited colony-forming uniterythroid (CFU-E) and CFU granulocyte-monocyte formation in BM culturesof PML+/+ and PML−/− progenitors. A detailed description of the methods,materials, and results of these experiments is provided in Wang et al.,Blood, 1998, 92:1497-1504.

Results from the experiments show that the cytotoxic effect of botharsenicals in these cell lines is not mediated by mechanisms that aredependent on PML or PML-RAR+ expression. In most lines, melarsoprol wassomewhat more potent compared with As₂O₃ in inhibiting growth andinducing apoptosis, and the effects of both drugs were dose dependent.As previously reported, it is confirmed that As₂O₃ relocalized PMLprotein onto nuclear bodies and induced PML and PML-RARα degradation inNB4 cells while triggering spoptosis. However, similar effects were alsoobserved in HL60 and U937 cells which do not harbor the PML-RARα fusiongene. Moreover, melarsoprol induced apoptosis in all the cell linestested without altering PML and/or PML-RARα.

The differentiating action of As₂O₃ and melarsoprol, appeared negligiblein vitro, and did not appear to depend on the expression and/ormodulation of PML and/or PML-RARα either. In fact, the small effectobserved by the inventors in long-term cultures (up to 2 weeks), wascomparable in all the cell lines tested with both compounds.

It is also found that bcl-2 downregulation, which has been previouslylinked to the antileukemic effects of As₂O₃ in APL, was also notdependent on expression of PML-RARα protein, because it occurred in theNB4 subclone 306 in which the intact protein is not detectable. Finally,to test whether PML expression was essential to the antileukemic effectsof arsenicals, both agents were tested in mouse embryonic fibroblastsand BM cells from animals wherein wild-type PML had been eliminated byhomologous recombination. In these cells wholly lacking PML expression,both As₂O₃ and melarsoprol were equally effective in inhibiting growthand inducing apoptosis, and both had similar effects on normal CFU-E andCFU-GM colony formation. Moreover, no differences between wild-type andPML−/− cells were observed. Without being limited by any theory,together, these data strongly support theory that the antileukemiceffects of these arsenicals occurs independently of both PML andPML-RARα expression. These results are in keeping with the medicinalhistory of arsenicals for diseases that are not characterized byalterations in PML protein such as, for instance, chronic myelocyticleukemia.

The results indicate that both As₂O₃ and melarsoprol are broadly activeas antileukemic agents in both myeloid and lymphoid diseases. Inconclusion, the data indicate that cytotoxic activity is not mediated bythe PML protein and therefore is not limited to diseases that areassociated with alterations in PML expression. Thus, the arseniccompounds of the invention have a potentially broader therapeutic rolethat is not confined to APL.

5.2. Clinical Study of Melarsoprol in Patients with Advanced Leukemia

Melarsoprol, an organic arsenical synthesized by complexing melarsenoxide with dimercaprol, has primarily been used for the treatment ofAfrican trypanosomiasis. The effects of melarsoprol upon induction ofapoptosis in cell lines representative of chronic B-celllymphoproliferative disorders have been investigated, and the resultsare described below.

Melarsoprol (supplied as Arsobal [36 mg/mL] by Rhone Poulenc Rorer,Collegeville, Pa.) was diluted in propylene glycol at a stockconcentration of 10-4 mol/L and stored at room temperature. As₂O₃(Sigma, St. Louis, Mo.) was dissolved in 1.65 mol/L sodium hydroxide(NaOH) at a stock solution of 10-3 mol/L. Serial dilution (10⁻⁶ to 10⁻⁹mol/L) were made in RPMI 1640 media. An Epstein-Barr virus(EBV)-transformed B-prolymphocytic cell line (JVM-2), an EBV-transformedB-cell chronic lymphocytic leukemia (B-CLL) cell line (I83CLL), and onenon-EBV-transformed B-CLL cell line (WSU-CLL) were used as targets.Dose-response experiments with melarsoprol (10⁻⁷ to 10⁻⁹ mol/L) wereperformed over 96 hours.

Unexpectedly, the inventors found that melarsoprol caused a dose- andtime-dependent inhibition of survival and growth in all three celllines. In contrast, As₂O₃ at similar concentrations had no effect oneither viability or growth. After 24 hours, all three cell lines treatedwith melarsoprol (10⁻⁷ mol/L) exhibited morphologic characteristics ofapoptosis. A prominent concentration-dependent downregulation of bcl-2mRNA after 24 hours of exposure to melarsoprol in WSU-CLL 183CLL, andJVM-2 cells was observed. Decrease of bcl-2 protein expression was alsoobserved in all three cell lines, whereas As₂O₃ had no effect on thisparameter.

Given that the in vitro data above have shown unexpectedly broadantileukemic activity for melarsoprol against both myeloid and lymphoidcells, and generally at lower concentrations than As₂O₃, a study wasinitiated to evaluate the pharmacokinetics, safety, and potentialefficacy of melarsoprol in human patients with relapsed or refractoryleukemia.

Eligible patients were treated with a brief IV injection daily for 3days, repeated weekly for 3 weeks, with an additional 3 wk course inresponding pts. The initial dose was 1 mg/kg on Day 1, 2 mg/kg on Day 2,and 3.6 mg/kg on Day 3 and all days thereafter. Parallel in vitrostudies included culture sensitivity of fresh leukemic cells to bothmelarsoprol and As₂O₃, along with serial flow cytometric studies ofsurface antigen expression, apoptosis, and bcl-2 expression. Threepatients with AML and one with CML have entered the study.

Using a method based on high performance liquid chromatography that issensitive to approximately 10 mg/ml, preliminary pharmacokinetic datashow that peak plasma drug concentrations were obtained immediatelyafter injection with a Cmax that ranged from 1.2 ng/ml on day 1 to 2.4ng/ml on day 3. While the initial distribution phase was rapid, aprolonged Thy has suggested release from a deep compartment. Plasmaareas under the concentration x time curves (AUCs) were proportional tothe administered dose, ranging from 0.48 ng·hr/ml on Day 1 to 1.48ng·hr/ml on Day 3. Detectable concentrations of the drug were found inplasma one week after initial dosing. The drug has been relativelywell-tolerated. Adverse effects have included transient pain at theinjection site and mild nausea. No signs of “reactive encephalopathy”(occasionally observed during treatment of CNS trypanosomiasis) havebeen observed.

Results from these studies suggest that melarsoprol may have broaderactivity than inorganic As₂O₃, and that concentrations which arecytotoxic to leukemic cells in vitro, and thus therapeutic, are readilyachieved in vivo.

5.3. Arsenic Trioxide Induces Apoptosis in K562 Chronic MyelogenousLeukemia (CML) Cells

A Philadelphia chromosome positive CML cell line K562 is used todetermine if arsenic trioxide (As₂O₃) promotes apoptosis in CML.Suspension cultures of cells in log phase were exposed to As₂O₃ atconcentrations of 1×10⁻⁵ M, 5×10⁻⁶ M, and 1×10⁻⁶ M. Aliquots of cellswere analyzed at various time points over the course of 72 hours toassess viability and apoptosis. Viability was measured using trypan blueexclusion; at the same time, apoptosis was detected by morphology, flowcytometry, and DNA gel electrophoresis.

Arsenic trioxide at a concentration of 1×10⁻⁶ M had no effect on K562cell growth or viability. The greatest effect on cell growth andsurvival was seen with 1×10⁻⁵ M As₂O₃ K562 cell growth and viabilitydata after 72 hours of exposure to As₂O₃ are recorded in Table 1:

TABLE 1 % Cell Growth Impairment % Viability p value control 0 92.1 ±0.9 5 × 10⁻⁶ M As₂O₃ 63.0 78.8 ± 0.5 0.0001 1 × 10⁻⁵ M As₂O₃ 75.3 61.9 ±2.9 0.0223

Evidence that this arsenic-induced decrease in viability representedapoptosis was analyzed. Morphologic features of apoptosis includingmembrane blebbing and nuclear condensation were evident in stainedcytospins of K562 cells incubated with 10⁻⁵ M As₂O₃ for 72 hours. Thiscorrelated with evidence of DNA internucleosomal damage as visualized bygel electrophoresis of DNA extracted from K562 cells exposed to 10⁻⁵ MAs₂O₃. Quantitative assessment of apoptosis, as measured by the TUNELmethod demonstrated that 75.6% ±8.6 (1×10⁻⁵ M As₂O₃) cells exhibitedapoptosis as compared with 6.3% ±3.0 (control) cells at 72 hours.Treatment of K562 cells with 10⁻⁵ M As₂O₃ resulted in an upregulation ofp21 mRNA, as detected by Northern analysis, suggesting an arrest of thecells in the G1 phase of the cell cycle. This data indicates arsenictrioxide as a therapeutic agent for CML.

5.4. Therapeutic Trials with Retinoic Acid and Arsenic Trioxide (As₂O₃)in PML-RARα AND PLZF-RARα Transgenic Mice

Acute promyelocytic leukemia (APL) is associated with chromosomaltranslocations which invariably involve the translocation of theRetinoic Acid Receptor α (RARα) locus on chromosome 17 to other loci inthe genome, such as in the majority of APL cases, the PML gene locatedon chromosome 15, and in a few cases the PLZF gene on chromosome 11.Patients harboring the t(15;17) are sensitive to treatment withAll-Trans Retinoic Acid (ATRA), yielding complete remission rates of 75%to 95%. APL associated with the t(11;17) (PLZF-RARα) shows a poorresponse to ATRA.

To test the efficacy of As₂O₃ in the treatment of APL, models of thedisease were created in transgenic mice. Transgenic mice were generatedby standard techniques in which the expression of the PML-RARα orPLZF-RARα fusion proteins is placed under the control of amyeloid-promyelocytic specific human Cathepsin-G (hCG) minigene. BothhCG-PML/RARα and hCG-PLZF-RARα transgenic mice develop myeloid leukemiawith features of APL similar to those in humans.

Therapeutic trials on these leukemic mice with the following regimenswere started: 1) ATRA: 1.5 μg per gram of body weight per dayadministered orally; and 2) ATRA: 6 μg per gram of body weight per dayadministered intraperitoneally. Mice were bled once a week to evaluatethe response.

PML/RARα leukemias responded well to ATRA with high remission rates (80%with regimen 1). Surprisingly, in vitro, ATRA induced differentiation,and inhibited growth of leukemic cells as well as leukemic colonyformation in bone marrow and spleen progenitors assays in both PML-RARαand PLZF-RARα leukemias. Furthermore, in ex vivo experiments, leukemiccells from PLZF-RARα mice lost their tumorigenic capacity whentransplanted in recipient nude mice upon pre-incubation with ATRA, whileuntreated cells were tumorigenic. However, in vivo, PLZF-RARα leukemiasresponded poorly to ATRA (28% with regimen 1), while higher doses ofATRA appeared more effective (50% with regimen 2). In conclusion,leukemias in PLZF-RARα transgenic mice are sensitive to ATRA treatment,but might require therapeutic regimens with high doses of ATRA. Thesefindings have direct implications in the treatment of APL patients witht(11;17).

In both PML-RARα and PLZF-RARα leukemias, ATRA prolonged survival, butleukemias relapsed shortly after remission was achieved, and wererefractory to further ATRA treatment. The two transgenic mouse models isalso used to test the efficacy and dosage of As₂O₃, and ATRA+As₂O₃ incombination for treatment of APL patients resistant to ATRA, and in APLassociated with the t(11;17). A regimen of As₂O₃ at 6 μg per day or acombination of As₂O₃ at 6 μg and ATRA at 1.5 or 6 μg per gram of bodyweight per day is administered intraperitoneally. Mice are bled weeklyto evaluate the remission of the APL.

5.5. Manufacture and Stability of Pharmaceutical Formulation

Solid ultrapure arsenic trioxide (As₂O₃) was solubilized in a solutionof 5 M sodium hydroxide (NaOH). The suspension was stirred at ambienttemperature for 5 minutes which yielded a clear, homogenous solution.The As₂O₃ solution (2 mL, 1.0 M) was added to 393.6 mL of H₂O in a 500ml Erlenmeyer flask, which yielded an As₂O₃ concentration of 1 mg/mL atpH=12. A 5.0 M HCl solution was prepared by dilution of HCl (49.26 mL,37% wt/wt, ¹⁰/₁₅ M) with H₂O (50.74 mL) in a 250 mL Erlenmeyer flask.The HCl solution was later transferred via syringe to a 1000 mL emptyevacuated container. The As₂O₃ solution was back titrated with HCl(0.725 mL, 5.0 M) to pH 8.0. Approximately 10 mL of the backtitratedAs₂O₃ solution was filtered through a Millex-GS 0.22 μm filter unit andwas added to each of approximately 30 sterile evacuated sterile vials.To make the pharmaceutical composition which would be injectedintravenously into patients, 10 mL of this solution was withdrawn fromtwo of the vials and was added to a 500 mL 5%-dextrose solution whichyielded a final pH of 6.5.

The high purity of the bulk starting material was confirmed (seeTable 1) by atomic absorptiometry. Duplicate samples of fourintermediate or final-step solutions were assayed for total arseniccontent. Assay bulk powder confirmed the extremely high purity of thestarting material. Data for arsenic content of the intermediate andfinished product solutions are presented in Table 2 below.

The data below show that the solutions are stable in that there does notappear to be any indication of weight loss of arsenic over time.

TABLE 2 Arsenic content (ppm) of intermediate formulation and finishedproduct solution of arsenic trioxide. Sample Code A-01* A-02 A-03 A-04A-05 Aliquot A 140,600 600 707 629 680 Aliquot B 139,000 564 703 688 687Assay 1.1% 6% 0.57% 8.7% Variance *Identity of sample codes: A-01:Intermediate product solution after initial solubilization in NaOH.A-02: Intermediate product solution prior to HCl titration. A-03:Intermediate product prior to Millex filtration. A-04: Finished productfrom sterile 10 ml fill vial immediately after manufacturing. A-05:Finished product from capped vials two months after manufacturing.

6. Examples Clinical Trials in Apl Patients

Arsenic trioxide was evaluated in patients with APL to determine whetherthis agent induced either cytodifferentiation or apoptosis. Twelvepatients who had relapsed from extensive prior therapy were treated witharsenic trioxide at doses ranging from 0.06 to 0.2 mg/kg per day until abone marrow remission was achieved. Bone marrow mononuclear cells wereserially monitored by flow cytometry for immunophenotype, fluorescencein situ hybridization (FISH), reverse transcription polymerase chainreaction (RT-PCR) assay for PML/RAR-α expression, and Western blotexpression of the apoptosis-associated proteins, caspases 1, 2 and 3.The results showed that low-doses of arsenic trioxide are highlyeffective for inducing complete remission in relapsed patients with APL.Clinical response is associated with incomplete cytodifferentiation andinduction of apoptosis with caspase activation in leukemic cells.

6.1. Methods

Clinical protocol: Eligibility requirements included a diagnosis of APLconfirmed by cytogenetics or fluorescence in situ hybridization (FISH)analysis for a t(15;17) translocation, or by reverse transcriptasepolymerase reaction (RT-PCR) assay for PML/RAR-α. Patients must haverelapsed from standard therapy that had included all-trans retinoic acidplus a combination of cytotoxic drugs. Signed informed consent wasrequired, and the protocol was reviewed and approved by this center'sinstitutional review board

Arsenic trioxide treatment: Arsenic trioxide was supplied as an aqueoussolution in 10 ml vials containing 1 mg/ml of drug. The drug was furtherdiluted in 500 ml of 5%-dextrose solution and infused intravenously over2 to 4 hours once per day. While the initial cohort of patients receivedeither 10 or 15 mg/day as a flat dose, the referral of two childrenprompted the invention of a weight-based regimen (0.15 mg/kg/day) thatwas heretofore unknown. The drug was given daily until bone marrowremission was observed. Patients who achieved complete remission wereeligible for treatment with additional courses of therapy 3 to 6 weeksafter the preceding course. Subsequent courses were generally given at adose of 0.15 mg/kg/day for a cumulative total of 25 days, administeredeither daily or on a weekdays-only schedule, for a maximum total of 6courses over approximately 10 months.

Monitoring during study: Patients with coagulopathy were transfused withplatelets and fresh-frozen plasma to maintain the platelet count andfibrinogen at target levels ≧50,000 cells/cu mm and ≧100 mg/dL,respectively. Blood counts, coagulation studies, serum chemistryprofiles, urinalyses, and electrocardiograms were serially obtained. Abone marrow aspiration and/or biopsy was performed at baseline andperiodically thereafter until remission was documented.

Conventional response criteria were observed, which included recovery ofbone marrow to ≦5% blasts, peripheral blood leukocytes ≧3,000 cells/cumm, and platelets ≧100,000 cells/cu mm.

Cellular immunophenotype studies: Heparinized bone marrow or bloodsamples were collected and mononuclear cells were isolated byFicoll-Hypaque centrifugation. Surface membrane antigens were detectedby direct immunofluorescence staining using fluorescein isethiocynate(FITC) or phycoerythrin conjugated monoclonal antibodies: CD16 (Leu11a), CD11b, CD33 (Leu M9), HLA-DR, CD45, and CD14, purchased fromeither Becton-Dickinson (Mountainview, Calif.) or Immunotech Immunology(Marseille, France). Dual-color staining was performed by incubatingcells simultaneously with two monoclonal antibodies, includingCD33-PE/CD11b-FITC and CD33-PE/CD16-FITC. Negative controls usingirrelevant monoclonal immunoglobulins of the same isotype were analyzedconcurrently. Flow cytometric analyses were performed on an EPICSProfile II flow cytometer (Coulter Electronics) equipped with a 488 nmargon laser. Forward and side-scatter cell parameters were measured andcombined with CD45/CD14 staining to identify populations of interest andto exclude monocytes from the analysis gate. The Multiparameter DataAcquisition and Display System (MDADS, Coulter Electronics) was used toacquire and analyze data.

Fluorescence in situ hybridization (FISH): Selected specimens that hadundergone immunofluorescence staining for CD33 and CD11b were sorted forcells that coexpressed both antigens using a FACStar Plus cell-sorter(Becton-Dickinson). Separated cells were incubated in culture media at37° C. for one hour, treated with hypotonic solution 0.075M KCl for 5minutes, fixed in 3:1 methanol:acetic acid fixative, and air-dried.Interphase FISH was performed using a specific PML/RAR-α translocationdual-color probe (Vysis; Downer's Grove, Ill.). Briefly, DNA frominterphase cells was denatured by immersing slides in a solution of 50%formamide/2×SSC at 73° C. for 5 minutes; the slides were then dehydratedin alcohol and air dried. A mixture of probe in hybridization mixturewas applied, covered with a cover slip, and sealed with rubber cement.Hybridization was carried out at 37° C. in a moist chamber forapproximately 12 to 16 hours. Following hybridization, unbound probe wasremoved by washing the slides at 45° C. in 50% formamide/2×SSC solutionthree times for 10 minutes each, followed by a wash in 2×SSC/0.1 NP-40solution at 45° C. for 5 minutes. Slides were then air dried andcounter-stained with 4′,6-diamidino-2-phenylindole and covered with aglass coverslip. Analysis of interphase cells for fluorescent signalswas performed with a Photometrics Sensys camera fitted to a Zeissaxioscope. A minimum of 300 cells was studied for each sample.

Western blot analysis: Cells were lysed in a buffer containing 50 mMTris-HCl, 0.5 mM ethylene glycol [bis]-[aminioacyl] tetra acetic acid,170 mM NaCl, 1 mM dithiothreitol, 0.2% NP-40, 0.01 U/mL aprotinin, 10μg/mL leupeptin, 10 μg/mL pepstatin, and 1 AM phenylmethylsulfonylfluoride (all from Sigma). The lysates were then sonicated using aultrasonic homogenizer (4710 series, Cole Parmer Instruments, Chicago,Ill.) and centrifuged at 7,500 g (Sorvall Instruments, Newtown, Conn.).Protein content of the lysates was determined using a BioRad ProteinAssay Kit (Bio-Rad Laboratories, Hercules, Calif.) at 595 nm with a BSAstandard. A sample buffer containing 10% glycerol, 0.4% SDS, 0.3%bromphenol blue, 0.2% pyronin Y, in 1× stacking buffer (Tris base 0.5 M,0.8% SDS), 20% 2-mercaptoethanol, was added to the cell lysates, whichwere heat-denatured at 95° C. for 3 min. Subsequently, 15 μg/lane ofprotein was loaded on a SDS-polyacrylamide gel containing 12.5%polyacrylamide and was size-fractionated by electrophoresis. Proteinswere electroblotted onto Tras-Blot® transfer medium (Bio-Rad) andstained with Ponceau-S as an internal loading control. Rabbit anti-humanmonoclonal antibodies, including caspase 1, caspase 2 (both from SantaCruz Biotechnology, Santa Cruz, Calif.), and caspase 3 (PharMingen, SanDiego, Calif.) were added, and bound antibodies were detected using theECL™ chemiluminescence detection system (Amersham, Arlington Heights,Ill.). Protein bands were quantified by computer densitometry.

RT-PCR analysis for PML/RAR-α: RT-PCR was performed using methodspreviously described (Miller et al., 1992, Proc. Natl. Acad. Sci.89:2694-8; Miller et al., 1993, Blood, 82:1689-94).

6.2. Results

Patients: Twelve patients with relapsed or refractory APL were treated.All patients had received extensive prior therapy with retinoids andcytotoxic drugs (Table 3). Two patients had relapsed from allogeneicbone marrow transplantation, one of whom had also failed donor T-cellreinfusion. One patient was being maintained on hemodialysis for chronicrenal failure.

Clinical Efficacy: Eleven of the 12 patients achieved a completeremission after arsenic trioxide treatment. The patient who entered onhemodialysis sustained an intracranial hemorrhage on day 1 and died onday 5. The median duration of therapy in responding patients was 33 days(range, 12 to 39 days), the median daily dose was 0.16 mg/kg (range,0.06 to 0.2 mg/kg), and the median cumulative dose during induction was360 mg (range, 160 to 515 mg) (Table 3). Complete remission by allcriteria was attained at a median time of 47 days (range, 24 to 83 days)after initiation of therapy. Remission by bone marrow criteri—thedetermining factor for discontinuing therapy—was achieved first, usuallyfollowed in sequence by recovery of peripheral blood leukocytes andplatelets. Over the range of doses used in this study, no differences inefficacy or time to response were obvious. After 2 courses of therapy, 8of 11 patients tested had converted their RT-PCR assays for PML/RAR-αfrom positive to negative.

All 11 patients in complete remission completed at least 1post-remission treatment course with arsenic trioxide. Four, two, andone patient each have completed a total of three, four and fivetreatment courses, respectively. The median duration of remission is5+months (range, 1 to 9+months). However, 3 of the 11 patients relapsedduring the second treatment course; none of these patients had convertedtheir RT-PCR assays, and each appeared to have rapidly acquired drugresistance. Two of these individuals have since expired from progressiveleukemia.

Adverse Events: The clinical condition of patients in this study washighly variable, which reflected their extensive prior therapy. Theprotocol did not require hospitalization; three patients completedinduction therapy entirely as outpatients, and one other individual washospitalized solely for placement of a venous catheter. However, 8patients were hospitalized for complications of leukemia, 5 of whomrequired transfer to an intensive care unit, endotracheal intubation,and assisted ventilation for complications that included pulmonaryhemorrhage, renal failure, sepsis, graft vs. host disease, non-specificpulmonary infiltrates, or hypotension. One patient required insertion ofa permanent pacemaker after second-degree heart block developed in thesetting of severe metabolic acidosis, hyperkalemia, hypotension, andrenal insufficiency. However, the heart block reversed despiterechallenge with further arsenic trioxide therapy. The drug wastemporarily withheld due to serious intercurrent medical complicationsin 5 patients for a median of 2 days (range, 1 to 5 days). Two patientsdeveloped symptoms similar to that of the “retinoic acid syndrome”; bothwere presumptively treated with dexamethasone and improved. Only 2patients required no platelet transfusions whatsoever; the median numberof platelet units transfused was 61 (range, 0 to 586 units).

The median total peripheral blood leukocyte count at entry was 4,700cells/cu mm (range, 500 to 144,000 cells/cu mm). Six patients developedleukocytosis (i.e., ≧20,000 cells/cu mm) that ranged from 20,800 to144,200 cells/cu mm. No additional therapy was administered to thesepatients, and the leukocytosis resolved in all cases without furtherintervention.

Common adverse reactions included lightheadedness during the infusion,fatigue, musculoskeletal pain, and mild hyperglycemia. Three patientsdeveloped dysesthias presumably due to peripheral neuropathy. However, 2of these patients had been immobilized for prolonged periods duringassisted ventilation, and the other patient had an antecedentneuropathic history.

Immunophenotype studies: APL is characterized by cells that expressCD33, an antigen typically associated with primitive myeloid cells.Arsenic trioxide therapy induced a progressive decrease in theproportion of cells that solely expressed CD33, along with an increasein the proportion of cells that expressed CD11b, an antigen associatedwith mature myeloid elements. While these changes would be anticipatedfrom any agent that induced remission in APL, arsenic trioxide alsoinduced expression of cells that simultaneously expressed both antigens.In most cases, these dual-expressing cells dominated the myeloid cellpopulation, and they persisted for extended periods after completeremission was achieved by clinical criteria.

Fluorescence in situ hybridization analysis: Bone marrow mononuclearcells taken from a patient both early and later in complete remissionwere sorted by flow cytometry for coexpression of CD33 and CD11b. Usingfluorescence in situ hybridization (FISH) analysis, three hundred cellswere examined early in remission. Similar to control APL cells, themajority of these cells yielded a hybrid signal, indicating atranslocation between PML and RAR-α genes and their origin from theneoplastic clone. However, when cells from the same patient were againsorted using these same parameters later in remission, only the normalpattern of fluorescence signals was detected, indicating theirderivation from normal hematopoietic progenitors.

Western blot analysis: Protein extracts from bone marrow mononuclearcells were serially examined by Western blot analysis. The analysisshowed that the precursor forms of caspase 2 and caspase 3 wereupregulated in vivo in response to arsenic trioxide treatment. Moreover,this treatment also induced expression of cleaved fragments of caspase1, indicating activation of the enzyme. There is also some indicationthat expression of the cleaved form of caspase 3 is increased. Theantibody used in these experiments does not react with the cleaved formof caspase 2.

6.3. Discussion

In this study, with few exceptions, patients admitted to the trial hadsustained multiple relapses and were resistant to conventionalchemotherapy, retinoids, or bone marrow transplantation. At entry,patients in this study suffered from numerous leukemia-relatedcomplications, including respiratory failure, disseminated Varicellazoster infection, cavitary aspergillosis, chronic renal failure, andgraft-vs.-host disease. Moreover, 5 of the 12 patients requiredadmission to an intensive care unit for assisted ventilation andsupportive care, but these complications were not directly related toarsenic trioxide therapy.

Virtually all patients with a confirmed diagnosis of APL attainedremission without the early mortality associated with retinoid therapy.Although less commonly observed compared with all-trans retinoic acidtreatment, arsenic trioxide induced striking leukocytosis-in severalpatients. Upon withholding other cytotoxic drugs, the leukocytosisdisappeared as patients attained remission. Despite 3 early relapses, 8of 11 patients tested converted RT-PCR assays for PML/RAR-α (a molecularmarker of residual disease) to negative, a phenomenon that is unusualafter all-trans retinoic acid treatment alone. Finally, arsenic trioxideis active in APL over at least a three-fold dose range from 0.06 to 0.20mg/kg.

All-trans retinoic acid induces “terminal” differentiation of APL cells,but the cytodifferentiating effects of arsenic trioxide appear to beincomplete. Arsenic induces a population of cells that simultaneouslyexpress surface antigens characteristic of both mature and immaturecells (i.e. CD11b and CD33, respectively). Early during induction, thesecells retain the t(15;17) translocation that characterizes APL.Unexpectedly, these cells persisted in the bone marrow despite theachievement of a clinically complete remission; however, later inremission, the coexpressing cells—while still readily detectable—were nolonger positive by in situ hybridization. The morphologic appearance ofleukemic cells during therapy is also far less distinctive than thatobserved during therapy with all-trans retinoic acid. In fact, leukemiccells from many patients displayed few morphologic changes for 10 ormore days, after which the proportion of leukemic cells progressivelydecreased.

Following “non-terminal” differentiation, arsenic trioxide appeared toinduce apoptosis, coincident with increased expression and conversion ofcysteine proteases (termed caspases) from inactive precursors toactivated enzymes. The caspase pathway has only recently beencharacterized as an important pathway of programmed cell death.Initially recognized due to homology between the C. elegans proteinced-3 and mammalian interleukin-1β converting enzyme (ICE), the familyof caspases now encompasses at least 10 different proteins that cleave anumber of polypeptides. In leukemic cell lines, caspase activation isinducible with a number of cytotoxic agents, including all-transretinoic acid. Since these enzymes induce widespread proteolysis, it isconceivable that PML/RAR-α is a caspase substrate.

A final similarity shared by arsenic trioxide and all-trans retinoicacid is the rapid development of clinical resistance in someindividuals. Leukemic cells taken from two patients who relapsedretained in vitro sensitivity over concentrations ranging from 10-4 M to10⁻⁷ M. Relative arsenic resistance due to decreased intracellulartransport has been described in association with down-regulation ofmembrane transporters encoded by the ars operon in bacterial cells.

Resistance in mammalian cells is less well-characterized, butalterations in membrane transport or efflux are probably importantfactors.

In summary, arsenic trioxide induces complete remission in patients withAPL who have relapsed from extensive prior therapy. This drug causespartial but incomplete cytodifferentiation of leukemic cells, followedby caspase activation and induction of apoptosis.

TABLE 3 Clinical characteristics and induction therapy results ofpatients with acute promyelocytic leukemia treated with arsenictrioxide. Treatment Time To Platelets Leukocytes Age No. of durationDaily Dose Cumulative Remission ≧100,000/ ≧3,000/ (yrs) Relapses (days)(mg/kg) Dose (mg) (days) cu mm cu mm 36 1* 36 .16 360 54 36 54 45 3*^(a)39 .12 390 83 39 83 31 3^(a,b) 37 .18 370 41 39 41 25 2 16 .06 160 24 1616 62 2*^(d) 30 .11 300 41 41 31 75 1 12 .20 180 30 30 30 40 1* 33 .16495 47 47 43 13 2*^(a,b) 27 .18 270 50 41 52  9 1* 33 .17 165 28 28 2870 1^(c) 28 .16 420 77 77 49 28 2* 36 .15 515 54 47 54 25 3  5 .15  75 †† † All patients had previously received one or more courses ofall-trans retinoic acid, plus an anthracycline antibiotic plus cytosinearabinoside. *Denotes individuals with proven retinoid resistance (i.e.lack of response during reinduction or relapse while on retinoidmaintenance); †Denotes patient who died early. Other treatment:^(a)mitoxantrone/etoposide; ^(b)allogeneic bone marrow transplantation;^(c)methotrexate/vincristine/6-mercaptopurine; ^(d)9-cis retinoic acidplus M195 (anti-CD33 monoclonal antibody).

7. Examples Clinical Use in Lymphoma

Based upon the initial discovery of the antitumor effects of arsenictrioxide in vitro against B-cell lymphocyte lines, the inventors treatedone patient with intermediate-grade large cell lymphoma who had relapsedfrom multiple forms of conventional therapy, including autologous bonemarrow transplantation. Despite rapid progression of his disease priorto starting the arsenic trioxide therapy, treatment with arsenictrioxide effected a major (>50%) shrinkage in the size of his cancerouslymph nodes and spleen, which was also associated with a majorimprovement of his quality of life.

8. Examples Clinical Use in Non-Hematopoietic Cancer

Arsenic trioxide was also used to treat colon cancer. In a preliminarytest, one patient with colon cancer who received one treatment witharsenic trioxide showed a major reduction in his serum CEA(carcinoembryonic antigen) level. The patient received daily intravenousinfusion of 0.1-5 mg arsenic trioxide per kg body weigh per day for fivedays. A change in the level of CEA from 19,901 ng/ml to 15,266 ng/ml, a23% reduction, was observed. It is well known that the a reduced levelof serum CEA is associated with antitumor response.

Clinical data confirms that arsenic trixoide can also be used to treatother non-hematopoietic cancer, such as colon cancer.

9. Examples Pharmacokinetics Studies

Several dose-ranging studies were conducted to examine thepharmacokinetics (PK) and biological effects of As₂O₃ in patients withAPL and in patients with other hematologic diseases. In patients withAPL, marrow mononuclear cells were serially monitored by flow cytometryfor immunophenotype, fluorescence in situ hybridization (FISH), andWestern blot expression of the apoptosis-associated proteins, caspases1, 2 and 3. Cells that coexpressed CD11b and CD33, and which by FISHanalysis carried the t(15;17) translocation, progressively increasedduring treatment and persisted early in complete remission. As₂O₃ alsoinduced in vivo expression of the proenzymes of caspase 2 and caspase 3,and activation of both caspase 1 and caspase 3. PK analysis of blood andurine for elemental arsenic (As) content showed that As was distributedin both plasma and red blood cell fractions of whole blood. Parallelelimination curves suggested that these 2 compartments were freelyexchangeable, and decayed from peak values with initial half lives ofabout 60 mins. The mean AUC on day 1 was about 400 ng·hr/ml.Approximately 20% of the administered dose was recovered in urine withinthe first 24 hrs.

We then initiated a dose-ranging study in patients with diseases otherthan APL using a daily intravenous dosing schedule for a cumulativetotal of 25 days per treatment course every 3-5 weeks at dose levels of0.1 and 0.15 mg per kg body weight per day. To date, 10 patients havebeen accrued, including patients with CLL (2 patients), AML (3patients), lymphoma (4 patients), and CML (1 patient). Five patientswere removed from the study early due to rapid progression, and 5completed the planned 25-day course. Over this dose range, the drug hasproved well-tolerated; adverse effects have included skin rash,lightheadedness during the infusion, fatigue, and QTc prolongation onEKG. Results from this ongoing study show that clinical use of As₂O₃induces partial differentiation and apoptosis in APL, but that thetherapeutic effects of this agent are not confined to this disorder.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A method for treating acute promyelocytic leukemia in a humancomprising administering a therapeutically effective dosage amount ofabout 0.15 mg/kg arsenic trioxide once per day.
 2. The method of claim1, wherein said arsenic trioxide is administered until bone marrowremission, which constitutes a first administration.
 3. The method ofclaim 2, further comprising a second administration of a therapeuticallyeffective dosage amount of about 0.15 mg/kg arsenic trioxide once perday for 25 doses.
 4. The method of claim 3, wherein said secondadministration is administered 3 to 6 weeks after said firstadministration.
 5. The method of claim 4, wherein said secondadministration is administered for up to five weeks.
 6. The method ofclaim 5, wherein said second administration is administered at fivedoses per week.
 7. The method of claim 3, further comprising repeatingsaid second administration.
 8. The method of claim 7, wherein saidsecond administration is repeated every 3 to 6 weeks.
 9. The method ofclaim 8, wherein said second administration is repeated until a total ofbetween two and ten cycles of said second administration are completed.10. The method of claim 9, further comprising repeating said secondadministration until a total of two cycles of said second administrationare completed.
 11. The method of claim 9, further comprising repeatingsaid second administration until a total of ten cycles of said secondadministration are completed.
 12. The method of claim 1, wherein saidarsenic trioxide is administered for up to sixty days, which constitutesa first administration.
 13. The method of claim 12, further comprising asecond administration of a therapeutically effective dosage amount ofabout 0.15 mg/kg arsenic trioxide once per day for 25 doses.
 14. Themethod of claim 13, wherein said second administration is administered 3to 6 weeks after said first administration.
 15. The method of claim 14,wherein said second administration is administered for up to five weeks.16. The method of claim 15, wherein said second administration isadministered at five doses per week.
 17. The method of claim 13, furthercomprising repeating said second administration.
 18. The method of claim17, wherein said second administration is repeated every 3 to 6 weeks.19. The method of claim 18, wherein said second administration isrepeated until a total of between two and ten cycles of said secondadministration are completed.
 20. The method of claim 19, furthercomprising repeating said second administration until a total of twocycles of said second administration are completed.
 21. The method ofclaim 19, further comprising repeating said second administration untila total of ten cycles of said second administration are completed.